Bottom Line:
We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR.Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system.Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.

ABSTRACTIn Alphaproteobacteria, the general stress response (GSR) is controlled by a conserved partner switch composed of the sigma factor σ(EcfG), its anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. Many species possess paralogues of one or several components of the system, but their roles remain largely elusive. Among Alphaproteobacteria that have been genome-sequenced so far, the genus Methylobacterium possesses the largest number of σ(EcfG) proteins. Here, we analyzed the six σ(EcfG) paralogues of Methylobacterium extorquens AM1. We show that these sigma factors are not truly redundant, but instead exhibit major and minor contributions to stress resistance and GSR target gene expression. We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR. Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system. Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.

pone.0152519.g007: Evolutionary relationships of σEcfG proteins of Methylobacterium species.The evolutionary history was inferred using the Neighbor-Joining method [26]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [27]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [28] and are in the units of the number of amino acid substitutions per site. The analysis involved 69 amino acid sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 171 positions in the final dataset.

Mentions:
Having analyzed the topology of the core cascade in M. extorquens AM1, we wondered whether σEcfG proteins were conserved in other Methylobacterium species. A search for ECF sigma factors in Methylobacterium species indicated they commonly harbor several ecfG genes (see S1 and S3 Tables). Phylogenetic analysis of σEcfG proteins of Methylobacterium species suggested that only σEcfG1 is conserved in all Methylobacterium strains considered (Fig 7). The other σEcfG proteins were only found in very closely related species, such as M. extorquens DM4, PA1 and CM4, and M. nodulans (Fig 7). Strains of more distantly related Methylobacterium species have their own set of σEcfG proteins, the number of which varies (Fig 7). Thus, in addition to σEcfG1, which is found in all species in agreement with its central role in the partner switch, Methylobacterium species possess different sets of σEcfG proteins, illustrating the plasticity of the system controlling the GSR in this alphaproteobacterial genus.

pone.0152519.g007: Evolutionary relationships of σEcfG proteins of Methylobacterium species.The evolutionary history was inferred using the Neighbor-Joining method [26]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [27]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [28] and are in the units of the number of amino acid substitutions per site. The analysis involved 69 amino acid sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 171 positions in the final dataset.

Mentions:
Having analyzed the topology of the core cascade in M. extorquens AM1, we wondered whether σEcfG proteins were conserved in other Methylobacterium species. A search for ECF sigma factors in Methylobacterium species indicated they commonly harbor several ecfG genes (see S1 and S3 Tables). Phylogenetic analysis of σEcfG proteins of Methylobacterium species suggested that only σEcfG1 is conserved in all Methylobacterium strains considered (Fig 7). The other σEcfG proteins were only found in very closely related species, such as M. extorquens DM4, PA1 and CM4, and M. nodulans (Fig 7). Strains of more distantly related Methylobacterium species have their own set of σEcfG proteins, the number of which varies (Fig 7). Thus, in addition to σEcfG1, which is found in all species in agreement with its central role in the partner switch, Methylobacterium species possess different sets of σEcfG proteins, illustrating the plasticity of the system controlling the GSR in this alphaproteobacterial genus.

Bottom Line:
We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR.Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system.Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.

ABSTRACTIn Alphaproteobacteria, the general stress response (GSR) is controlled by a conserved partner switch composed of the sigma factor σ(EcfG), its anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. Many species possess paralogues of one or several components of the system, but their roles remain largely elusive. Among Alphaproteobacteria that have been genome-sequenced so far, the genus Methylobacterium possesses the largest number of σ(EcfG) proteins. Here, we analyzed the six σ(EcfG) paralogues of Methylobacterium extorquens AM1. We show that these sigma factors are not truly redundant, but instead exhibit major and minor contributions to stress resistance and GSR target gene expression. We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR. Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system. Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.